Device for the simultaneous dialysis of a number of fluid samples

ABSTRACT

The invention elates to a device for the simultaneous dialysis of a number of fluid samples, comprising a vessel for the dialysis fluid, with an inlet and outlet, a fill-level regulation and a sample plate ( 3 ) with a number of similar sample vessels ( 5 ) of μl range size, arranged in a raster (n×8×12), the upper ends of which are open and the lower ends of which are sealed by a semi-permeable membrane ( 7 ) lying in a plane. In the raster region of the sample vessels ( 5 ), the sample plate has no regions or elements which extend beyond the plane of the membrane ( 7 ), or which form or support gas barriers after dipping in the dialysis fluid ( 2 ). The sample plate ( 3 ) comprises elements in the boundary region thereof for the escape of air after dipping in the dialysis fluid. Both sample fluids and dialysis fluids are agitated.

DESCRIPTION

The invention concerns an apparatus for the simultaneous dialysis of aplurality of liquid samples, which are contained in sample wells in asample plate, and have been brought to this sample plate for a dialysisseparation by at lest one semipermeable membrane which is in contactwith a dialysate. Such a dialysis system can find general applicationwhere, for analytical purposes, specifically a plurality of liquidmicrosamples is to be investigated regarding their the macro-molecularapportionment and from which microsamples, low molecular weightmolecules, which would disturb the analysis, must be separated. Theseparation is to be carried out in an efficient, easily manipulatedmanner and at the lowest possible expense. Beyond this, the dialysissystem is to be applied advantageously in order to concentratemacromolecular containing microsamples quickly, protectively and withoutsubstantial loss. A further area of application is the buffering ofsamples, especially in the range of the DNA-treatment, for theinvestigation of proteins and in the case of sequentially occurringenzymatic reactions.

In recent years, highly parallel screening techniques, such as HighThroughput Screening=HTS and Ultra High Throughput Screening=UHTS, havebeen showing up as analytical methods very frequently in analyticalwork. This has been especially aided by the efforts of thepharmaceutical industry for the capture of targets for the developmentof newer pharmaceuticals. Also, the proteomanic analysis is vigorouslydeveloping itself, and enables a very high sample throughput for thecharacterization of a multiplicity of proteins of a proteome withbiological modifications both in various conditions, such a healthy andill. Beyond this, the said proteomic analysis is an indispensable aidfor many application ranges in biochemical and biotechnical researchwith high throughput analyses, for example, for the characterization ofenzymes in regard to their activity, for the characterization ofanalytical and preparative chromatographic separations, for the masscentered renaturation of protein samples and for the characterization ofnucleic acids.

These high throughput procedures have led, in their general range, toacceptable auxiliary technologies for the development of specialanalysis procedures. For example, these group themselves around amicrotiter of 8×12 analysis positions in the basic area of a well plateof about 12.6×8.6 cm. At the present time, attention is given to a trendfor even further compression of the analysis positions on the same basicarea of n×8×12, where n can equal 4, or even 16 and further to moredensely compacted multiple well plates. Very frequently, the materialsof interest can be macromolecular substances such as DNA and itsfractionates, proteins, peptides, glycoprotein and syntheticmacromolecules as well as combinations of these substances.

Two additional and new procedures, MALDI-MS (Matrix Assisted LaserDesorption Ionization Mass Spectrometry) and ESI-MS (ElectrosprayIonization Mass Spectrometry) have been recently developed in the lastfew years, and have proved themselves as excellent procedures forHTS/UHTS and for the proteomic analysis. In this case, especially incombination with protease digestion methods. In general, a strongtendency toward miniaturization of the methods of analysis must berecognized. Many procedures for the high-throughput screening methodshow a strong demand for sample preparation with high requirements. Someof the requirements are listed below:

-   -   1. the samples may contain only a low concentration of salt or        detergent, or the samples must be found in a specific millieu of        ionization,    -   2. the samples, which lie in the μl-volume range, must be        treated in a highly parallelized manner, in order to guarantee        the required analysis frequency, The treatment must be carried        out, uniformly and under standardized conditions for all        samples,    -   3. the recovery for these microsamples in the process of the        sample preparation, must be satisfactorily high, and    -   4. frequently, samples of biological material must be        protectively concentrated before the analysis, in order to        achieve the necessary desired level of concentration.

For the removal of low-molecular substances, for the transfer of thesamples into a defined millieu and for the concentration ofmacromolecular materials, excellent results have been acquired with thedialysis procedure requiring the use of semi permeable membranes.Consequently, there has arisen a greater number firms offering dialysisprocedures, which, for example, can be found collected together underthe address of http://biosupplynet.com.

The principal effort of the firms and the inventors for the improvementof the dialysis technology, addresses those problems, which relate tothe practical manipulation of the technology. Some of the problems to beovercome are: mixing, recovery of macromolecules, difficulties with thespeed of the dialysis process, and operations which are connected withsmall volumes, i.e., in the μl-range.

Where the MALDI and the ESI-MS procedures are concerned, the presentstate of the technology finds the removal of salts, detergents and othercontaminating substances necessary.

Possibilities, which could improve the quality of the essential mixingof a dialysis solution, are described in U.S. Pat. No. 5,183,564 and inU.S. Pat. No. 6,176,609. U.S. Pat. No. 6,176,609 in particular describesa general procedure for mixing in a plurality of vessels. Givingconsideration to the universal trend for miniaturization, a series ofsolutions were suggested. Also, U.S. Pat. No. 6,039,871 teaches ofequipment which can be looked at as being disposable and the dialysis of10 to 100 μl samples is foreseen as being in special vessels which canbe encapsulated, one in the other. In U.S. Pat. No. 5,733,442 a closedmicrodialysis system is proposed, which is marked by microchambers,which can be screwed together, and possess two dialysis membranes and aspecial stirring device.

The difficulties which arise in connection with recovery of smallanalyte volumes is given attention by the inclusion of a special capturechamber in U.S. Pat. No. 6,217,772.

In the documents U.S. Pat. No. 5,783,075 and U.S. Pat. No. 5,503,741 aproposal for floating dialysis in vessels of a special configuration ismade known. Where the floating dialysis is concerned, there are alsoofferings from the firm PGC, the firm Daigger and the firm Pierce, wherethe widely publicized slide dialysis system is treated.

In order to increase the rapidity of the dialysis, giving considerationto the ESI-MS technology, a capillary dialysis system is taught by U.S.Pat. No. 5,954,959.

Where a multiplicity of samples must be treated, proposals are put forthin U.S. Pat. No. 4,450,076. In these proposals the dialysis vessel isplaced about a central axle, and is turned by means of rotation. Offersfrom PGC Scientifics Corp. bring forward a central axis oriented,equilibrium chamber, which is sealed by Teflon coated screws. PierceBiotechnology, Inc. offers a microdialysis system for 12×20−100 μlvolumes.

The proposed solutions of the problem, however, are not adaptable to thedemands of higher throughputs, such as are necessary for HTS, UHTS andfor proteomic analysis because of the following, because they:

-   -   1. are not acceptable for highly parallel microplate technology        especially where corresponding liquid treatment is necessary,    -   2. a simultaneous and essentially uniform dialysis executed for        all samples in a greater sample count in the μl range is        prohibited,    -   3. a satisfactorily sufficient recovery of these small volumes        fro secondary analytical procedures is not allowable,    -   4. a correspondingly high dialysis speed is not provided,    -   5. the volumes required for the dialysate are inadequately        large, or    -   6. because of their complicated manner of application, the said        solutions are not practical in routine operation with a high        throughput.

Thus, the invention has the purpose, of dialysing simultaneously amultiplicity of micro samples in the μl-range essentially uniformly,wherein the manipulation is easily carried out and at as low a cost aspossible, whereby these dialyses can be executed quickly and, ifrequired, in an automatized manner, within the requirements of the modemscreen and analysis methods.

In the case of a large throughput of samples, the dialysis permits asufficiently high recovery of small volumes for secondary analyticalprocedures.

In accord with the purpose of the present invention, there is proposedan apparatus for the simultaneous dialysis of a plurality of liquidsamples, wherein:

-   -   there is included a dialysis vessel with a dialysate as well as        means for the inflow and outflow of the dialysate, which remain        in connection with a level control,    -   there is included at least one sample plate, which is either        immersed in, or is in contact with, the surface of the        dialysate, as well as being held by holding elements, in or on        the surface of the dialysate, with a matrix (n×8×12) arrangement        of a plurality of equal sample wells, which arrangement is        acceptable and known for liquid handling technique in microplate        technology and said wells have holding capacities for microliter        volumes, whereby, of the sample wells, respectively, the upper        ends are open and the lower ends are closed by a semipermeable        membrane lying in a plane, and the sample plate in the area of        the said matrix of the matrix of the sample wells has no gas        barrier forming or supporting zones or elements which extend        beyond the plane of the semipermeable membrane and the said        sample plate 3 has, in its rim areas, elements, such as openings        for the release of air imprisoned by touching contact with the        dialysate,    -   and besides the above, the said apparatus contains means for the        movement of the plate and the dialysate.

Enabled by the acceptability of the sample plate used in the dialysissystem, for the said liquid handling technique for the microplatetechnology of liquid, samples can be prepared with a high degree ofthroughput in accord with this technology, then subsequently be employedfor their purpose and finally recovered for further use. With thespecial design of the sample plate, those air barriers which obstructthe dialysis upon the immersion of, or the displacement of the plateinto or onto the dialysate surface are avoided. Should procedurallyevolved gases migrate into the contact zone between the dialysate andthe sample plate, then these can be forced out by deaeratingapparatuses, so that in any case, an unbroken contact can be assuredbetween the dialysate and the sample plate without the said gasbarriers, such as air bubbles and the like. This disturbance-freecontact is an essential presupposition for simultaneous and essentiallyuniform dialysis for each of the large number of samples in the μlrange. This effect is essentially supported by the movement of thesample liquids and the dialysate, so that, predominately, no secondarymembrane formation between the dialysate and the sample volumes can formand the dialysis can continue with unbroken continuity. To serve thispurpose, the dialysis vessel possesses at least one entry and one exitopening, in order that the materials which are accumulated in thedialysate can again be continuously expelled from the said dialysate andcontinually a dialysate with equal dialysis acceptance power remainsavailable in the system. The entry and exit flows are, meanwhile,connected by a level control, in order to hold constant the conditionsof dialysis on the semipermeable contact between the sample liquid andthe dialysate, thus maintaining the above advantages with considerationfor level control.

By means of the movement of not only the dialysate, but also of thesample liquid, this movement being done by a known shaking device, withwhich the sample plate is connected, not only is the high dialysiseffectivity itself attained, but also special usages, notablydialysis-effectivity is achieved. Of the latter, a dialysis ofdetergents which form micellla, is already enabled.

With the above stated features, the realization of variously designeddialysis systems is enabled, wherein the said dialysis systems can carryout different applications, either manually or with automatic drive,these being independent therefrom, as to whether the sample plate, forinstance, by means of a pivot or a shaking arm lies on the dialysisvessel or floats in the dialysate which is present in the container.

In the most simple case, without limiting the invention, the sampleplate may consist of a plate with cylindrical recesses or wells in thereceiving means for microliter volumes. On the underside of the sampleplate are the borings (sample containing recesses) either respectivelyclosed by a common or by individual dialysis membranes, which, forexample, are adhesively held on the underside of the plate. They mayalso be welded, bonded, or sprayed on. The dialysis membranes can alsoconsist of more than one layer.

A cover or an adhesive film of a releasable closure of the upper end ofthe sample vessel protects the sample material which is in the samplewells, and blocks any evaporation and contamination of the smallquantity of sample in the microliter range.

Described and explained in the subordinate claims, are a multitude ofadvantageous embodiments of the invented features. In this way, thedialysis vessel with the accepted sample plate becomes an integral partof a circulation system. In such a circulation system, for example, witha pump controlled, recycling apparatus, it is possible that anion-exchange device or a detergent capturing adsorber could be placed.Such an addition would hold the concentration of the substances whichare to be removed from the dialysate to a very small level, and therebythe speed of the dialysis procedure would be increased and the necessaryvolumes of the dialysate would be simultaneously minimized. Also the useof bound substances which form complex substances is possible, in orderto remove metal ions.

In the following, the invention is described and explained with the aidof embodiments as shown in the drawing. There is shown in:

FIG. 1: A sample plate, secured at the base of a dialysis vessel byfeet,

FIG. 2: An apparatus with a floating holder of the sample plate on thesurface of the dialysate,

FIG. 3: A dialysis vessel with the holder of the sample plate in accordwith FIG. 1, with both entry and exit fittings for the dialysate.

FIG. 4: A dialysis vessel with a sample plate in a circulation systemfor the removal of interfering substances from the dialysate,

FIG. 5: An apparatus for dialysis, wherein the sample plate is held in ashaking device to create turbulence in said dialysis apparatus,

FIG. 6: A floating holding means for the sample plate (see FIG. 2) withconical sample wells,

FIG. 7: A sequential run of a procedure to transfer dialyzed samplematerial subjected to centrifugation out of the sample plate withconical sample wells into a receiving plate with cylindrical wells,

FIG. 8: A second sequential run of a procedure similar to FIG. 7,showing a sealing means between the sample plate and the receivingplate, and

FIG. 9: A graph showing the conductivity of the dialysate during aperiod of dialysis.

In FIG. 1 is shown a dialysis container 1 with a dialysate 2 therein. Onthe bottom of, and within the dialysis container, is to be found asample plate 3, which is secured by a holder 4. This sample plate 3consists of a plate shaped, basic body in which, and within thespecifications of a known liquid handling technique for acceptablemicroplate technology, is placed an 8×12 matrix of aligned cylindricalwells 5 for the acceptance of sample material 6, the content of eachwell being in the microliter range.

On the underside of the sample late 3 is found a dialysis membrane 7,this membrane being semipermeable, common to all sample wells andsecured on the rims thereof by adhesive. By means of this dialysismembrane 7, each individual portion of the sample material 6, which iswithin the wells 5, stands respectively in contact with the dialysate 2.For this purpose, the sample plate 3 is so supported by the holder 4,that the said plate 4 is immersed, with its dialysis membrane 7, intothe dialysate. By means of the dialysis membrane 7, the exchange ofsmall molecules is possible, in accord with the exclusion threshold,since a concentration equilibrium between the dialysate 2 and theliquids of the sample material 6 is in force. The removal of the saidsmall molecules out of the material 6 of the samples is accomplished bythe effort of the said solutions to establish the mentioned equilibriumbetween the two compartments. Large molecules are restrained frompassing through the dialysis membrane 7.

The plane of the dialysis membrane 7 incorporates, in a way, also thelowest level of the sample plate 3, during its operation in accord withits application. There exists in this matrix area of the sample wells 5no zones or elements for stabilization fastening, manipulation or thelike, or even yet areas dependent upon fabrication, which would protrudefrom the sample plate 3 outward and beyond, which would contactinglyimpinge on the dialysate 2, or be immersed therein. Further thearrangement is such that no extension of the said zones or elementsexist, which would interfere with the uniformly running dialysis processby introducing air or creating air barriers.

By means of a known magnetic stirrer 8 on the bottom of the dialysiscontainer 1, the dialysate is held in motion, in order that aconcentration gradient on the dialysis membrane 7 be held as small aspossible and also to accelerate the dialysis. An adhesive foil 9,serving as a releasable closure of the upper rims of the sample wells 5,protects the sample material 6 which is found therein and prevents anevaporation or a contamination of the sample very small volumes.

FIG. 2 depicts a construction, which is very similar to that of FIG. 1.The difference, in this case, is that the sample plate 3 is notsupported by foot or structural holding elements 4 rigidly connected tothe bottom of the dialysis container 1, but is held by means of aframelike, float element 10 directly on the surface of the dialysate.This is accomplished in such a manner, that the dialysis membrane 7 lieson this surface. This mode of holding is independent of the level of thedialysate 2 in the dialysis container 1.

Additionally, the sample plate 3 has air escape openings 11, which allowgas collecting under the sample plate 3 to bleed out, thereby assuringan unbroken contact of the dialysis membrane 7 with the surface of thedialysate 2. This complete contact coverage forms the necessarypreparation for a simultaneous and essentially uniformly completeddialysis for each sample of this large number of samples in theμl-volume range.

Because the sample plate 3, at least in the matrix area of the samplewells 5, possesses no elements (for holding or the like), which wouldprotrude downward beneath the plane of the dialysis membrane 7 and thusimmerse themselves in the dialysate 2 or cause turbulence in the same,either of which would disturb the desired uniformity of simultaneousanalyses, very quickly essential characteristics for value-determiningusage of the sample plate 3 in a dialysis system were immediately takenadvantage of, in order to avoid air locks, or at least not to supportthem, in the contact zone of the dialysate 2 against the sample plate 3.If, nevertheless, gases evolved from processing appeared in this zone,then, these gases, as mentioned above, could disperse through the airescape openings 11 and emerge above trough the sample plate 3. Insteadof the air escape openings 11, other gas dispersing elements were givenconsideration, such as edge phase-changing, or the like. To enhanceclarity, details of the air escape openings 11 (or other deaerationequipment) were not explicitly shown in each figure presentation.

FIG. 3 shows an apparatus for dialysis, wherein the sample plate 3 (asin FIG. 1) is supported in the interior of the dialysis container 1 onthe floor thereof by means of feet or standard holding devices 4. Thedialysis container 1 possesses in this case, a feed fitting 12 as wellas a outlet fitting 13 for the dialysate 2. The level of the dialysate 2is regulated by means of an adjustable float 14 with a float actuatedvalve 15. In this case, the float 14 is guided to be vertically movablein a float track 16. The advantage of this, is to be found in thecontinuous content balancing of the dialysate 2. In this way, theadjustment of a concentration equilibrium between the dialysate 2 andthe respective sample material 6 contained in the wells 5 can beavoided, also the speed of the dialysis is greater and the removal oflow molecular substances from the sample material 6 is fundamentallyimproved. Naturally, the float valve system (12-17) can be replaced byan electronic level controller which regulates the feed at inlet 12.

FIG. 4 shows an apparatus for dialysis, wherein the dialysis container1, which is shown in top view, demonstrates the therein placed sampleplate 3 (see FIGS. 1 and 3), and shows the inlet 12 as well as theoutlet 13 as being components of the circulation system for thethrough-flow of the dialysate 2. The exchange of the dialysate 2 notcarried out, as in the apparatus of FIG. 3, through an open system, butrather by the dialysate 2 being transported by a pump 17, through afilter cartridge 18 and into a line 19, with the circulation systembeing completed by passage through the dialysis container 1. Thedirection of the dialysate 2 is indicated by an arrow, whereby the blackarrows symbolize the exit flow of the dialysate 2. Upon its exit out ofthe dialysis container 1, the dialysate 2, for example, can be enrichedwith ions and/or detergents from the sample material 6. By means of oneor more sorbents in the filter cartridge 18, these components can beremoved from circulation. The cleaned dialysate 2 thus migrates, as thewhite arrows show, back into the dialysis container 1. Advantageously,here, the ubiquitous applicability of the dialysate 2 contributes to:

-   (a) the said avoidance of the adjustment of a concentration    equilibrium, between the dialysate 2 and the sample material 6 held    respectively in the sample wells 5 and-   (b) the thereto connected advantages for dialysis (see the    embodiment example of FIG. 3).

FIG. 5 depicts an apparatus for dialysis (once again sectional profileand top views), wherein the sample plate 3 is neither anchored to thebottom of the dialysis container 1 (see FIG. 1) nor is it floating onthe dialysate 2 surface in the said dialysis container 1 (see FIG. 2).Rather the sample plate 3 is held by a holder 20, which is alsoconnected to a shaking device 22 through a shaker arm 21. The shaker 22serves, as the white, crossed arrows indicate, for the horizontalmovement of the sample plate 3 along the surface of the dialysate 2 andalso moves the sample plate 3 within the amplitude and frequency limitsas directed for a shaker installed for microtiter sample plates as theselimits are defined for laboratory operation. In this way, the samplematerial 6 found in the sample wells 5 of the sample plate 3 isthoroughly mixed, which acts against the establishment of concentrationgradients in the said sample material 6, as well as in the dialysate 2,which the shaker 8 also affects. This has the favorable advantage, thatthe somewhat hindering construction of secondary membranes, which arenecessary for many dialysis processes, may be omitted. Also, thetransporting away of gas bubbles in the area of the membrane is favoredby this shaking motion.

The advantage of this apparatus is, that not only is the recirculationand cleaning associated with the content balance of the dialysate 2, asis described for the embodiments of FIG. 3, 4, omitted, but also thespeed and completeness of the dialysis is improved. To preserve clarity,a combined presentation with the said, and previously describedembodiments, is not specifically illustrated in the attached drawings.In an additional embodiment example, not shown here, the motion of thesamples and dialysate can be carried out with the same positive effectsalso by being coupled with an ultrasonic mixer.

FIG. 6 shows (likewise in sectional profile and top views) an apparatusfor dialysis, wherein the sample plate 3, as is the case in FIG. 2,floats on the surface of the dialysate 2. The sample plate 3 does notpossess, as was the case in the previously described embodiments, samplewells with cylindrically parallel walls, but sample wells 5 a which areconically tapered, in the form of a cone frustum with respectivelylarger lower openings, which are closed by the dialysis membrane 7. Thesaid wells 5 a have, in comparison to the lower openings, smaller upperopenings. The smaller upper openings create an advantageousshape-closure (see FIG. 7) for the transfer of the sample material 6after the dialysis (following the dialysis) into the individual wellvolumes, in the same matrix in another sample receiver plate. Thisprocedure is in accord with known microtiter plate technology. Easilyrecognizable in the top view presentation of FIG. 6 is adhesive foil 9over the matrix arrangement of the sample wells 5 a, which foil preventsevaporation, spilling, and contamination of the sample material 6, whichis in the wells 5 a during the dialysis or can occur even duringtransport.

FIGS. 7, 8 show, schematically, respectively in sectional views throughthe plate, a sequential run of the procedure through centrifugation, outof the sample plate 3 with conical sample wells (see FIG. 6) into areceiver plate 23 with cylindrical wells. The receiving plate 23, in anupset position, is placed on top of the sample plate 3, whereupon thetwo are turned over in common. By means of centrifugation, the samplematerial 6, which is originally in the sample plate 3 is transferred tothe receiver plate 23. Subsequently, the sample plate 3 and the receiverplate 23 are taken apart. As a centrifugal device, the known laboratorycentrifuge for microtiter plates can be used. In FIG. 7, the upperopenings of the wells 5 are smaller than the openings of the samplecontainer of the receiver plate 23 which confronts them. On thisaccount, the already described satisfactory shape-fit assures thepenetrative interconnection as shown in the drawing 7. Conversely, inFIG. 8, we see the confronting well openings of the sample plate 3 andthe receiving plate 23 respectively equal in size. In this case, arequired tighter shape fit is assured by means of an intermediatelyinserted sealing means 24 between the sample plate 3 and the sampleplate 23.

In the following four embodiments is shown, how, with the describedapparatuses, different substances can be dialyzed.

APPLICATION EXAMPLE NO. 1

Low-molecular substances in 96 samples such as p-nitrophenol (p-NP) andsodium chloride, are to be uniformly removed, in short dialysis periods,wherein the reception of the sample plate 3 in a shaking device (seeFIG. 5) is provided and which said plate 3, in accord with FIG. 4, isconnected into a circulating system for the dialysate 2.

For this situation, into the 8×12 dialysis wells, which are closed witha VSMP Millipore membrane (0.025 μm), each 100 μl of a 1.5 mM p-NPsolution in 50-mM diethanol-amine buffer pH=9.8 (DEA), which, inaddition, contains 750 mM NaCl, is pipetted and dialyzed against avolume, which is only 11 times greater than a volume of 110 ml deionizedwater for 2 hours. The dialysate 2 is circulated by a hose pump. In thecircuit is integrated a deionizing column (Eco Pac, 10 ml, Bio Rad) (seeFIG. 4). During the dialysis, the sample plate 3 is continually shaken,which sample plate is closed with adhesive foil 9 and is fastened in theholder 20 of the shaping apparatus (see FIG. 5). The effectivity of theseparation of the lower molecular nitrophenols following the dialysis ischecked with the absorbencies of the outlet solution. The absorbencymeasurement of the outlet solution, which emerges from the dialysis, iscarried out with a DEA-buffer solution, pH=9.8 in a microtiter plate andthe absorbencies read off on a display. For the absorbency measurementof the 96 dialyzed samples, 3 aliquots are taken by pipette from thesample plate 3, with 50 mM DEA-buffer solution pH=9.8, mixed in amicrotiter plate and measured in a display.

The comparison of the absorbency in the 96 positions of the dialysismodule is shown in Table 1. The measured absorbencies have been reducedby the blind buffer value. TABLE 1 Comparison of the Absorbencies at 405nm (A₄₀₅) before and after Dialysis A₄₀₅ Analysis Dilution SolutionFactor Av. Val. Analysis Samples (n = 96) Solution A₄₀₅ Total p-NP(%)p-NP pre Dialysis 0.397 ± 0.005 30 11.910 100 p-NP post Dialysis 0.028 ±0.002 3.75 0.105 0.88

The values indicate, that under the described conditions, more than 99%of the p-NP can be removed. The distribution of the absorbency valuesafter the dialysis show, that the dialysis speed in all 96 dialysiswells is very much the same. Besides the comparison of the absorbenciesbefore and after the dialysis, the conductivities of the employedsamples were measured and were compared with the conductivities afterthe dialysis of the 96 dialysis wells. From these values a residualcapability of conductivity was determined, in relation to the outletsolution of 0.2%, which, in any case, confirms the effectivity of thedialysis.

APPLICATION EXAMPLE NO. 2

For the removal of lower molecular ions from proteins, in 8×11 positionsof the sample plate 3, per well, 75 μl of concentrated solution of analkaline phosphatase (14.5 μg/ml) was added to IM DEA buffer pH=9.8, anddialyzed for one hour against a large volume of 470 ml deionized water.The sample plate 3, which is placed as a floating element 10 on apolysterol framing, and floats on the dialysate 2, which is kept inmotion by the magnetic stirrer 8 (see FIG. 2). The continuous removal ofthe low molecular substances in the sample wells 5 is monitored by themeasurement of the conductivity (see FIG. 9) in the said dialysate 2.

After 60 minutes of dialyzation, 60% of the ions which can be dialyzedhave been removed. The determination of the enzymatic activities of thealkaline phosphatase in the 88 occupied dialyzation wells of the sampleplate 3 and a comparison of the enzymatic activity with the outletsolution yielded a recovery of the enzymatic activity of 90.4+4.3%.

APPLICATION EXAMPLE NO. 3

On the example of Triton X-100 (TX-100), the point is to determine, ifit is possible to remove this much used detergent by dialysis fromanalysis samples. To this end the sample plate 3 is, respective bywells, charged with 7511 of a 0.5% aqueous solution of Triton X-100 anddialyzed for 8 hours vs tap water. The sample plate 3 was closed byadhesive foil 9, and again placed in the holder 20 of the shakerapparatus (see FIG. 5) for continuous agitation. During this part of theoperation, the same was immersed in 170 ml of tap water as a dialysate2, which was renewed at flow rate of 170 Ml/min in a circulation systemin accord with FIG. 4.

In order to capture eventual volume changes, the sample plate 3 wasweighed before and after the dialysis. For the determination of theeffectivity of the removal of the Triton by dialysis, aliquots weretaken by multipipettes from the dialysis containers of the module, mixedin a microtiter plate with 30% n-propanol and measured in a fluorescencedisplay at an excitation wave length of 270 nm from an emission wavelength of 310 nm. The Triton X-100 which was subjected to dialyzation,after dilution in 30% n-propanol was measured under the same conditionsin a microtiter plate. For the correction of the measured values and forthe regulation of the linearity of the range of measurement, both thepropanol solution in 56 positions of the microtiter plate (blind values)and three standard concentrations of Triton X-100, between 12.5 and 50μM under the same conditions were measured. The results are summarizedin Table 2. The measured average values were reduced by the determinedpropanol blind value: Dilution Fluorescent Factor Analysis SolutionAnalysis Fluorescence Samples Av. Val. (n = 96) Solution Total TX-100(%) TX-100 739.6 ± 11.83 201 148 660 100 pre Dialysis TX-100 62.39 ±6.24   30  1 872 1.26 post DialysisThe measured fluorescences following the dialysis in the 96 dialysiswells of the sample plate 3 show, that under the described conditions,98.7% of the Triton X-100 was uniformly removed from all 96 of thedialysis wells.

APPLICATION EXAMPLE NO. 4

The sample plate 3 can also be employed for the simultaneousconcentration of 96 samples. For this purpose, in accord with each well,100 μl of a 0.3% Dextra-blueing solution with a multi-pipette was placedin the sample wells 5 of the sample plate 3. The sample plate 3 wassubsequently fixed on the floating frames 10 (see FIG. 2) of polysterol,which were laid on 100 ml of a 30% aqueous polyethylene glycol solution(PEG 40 000). After 45 minutes, the volume reduction was quantitativelydetermined. To this, from 88 out of the 96 positions was taken, perposition, 30 μl with a multi-pipette and transferred into a microtiterplate, which, per container, contained 120 μl 50 mM DEA-buffer solution.In the remaining 8 positions of the microtiter plate, instead of thesample solution, each was given 30 μl reference solution (0.3%Dextra-blueing solution). The microtiter plate was then, after intensivemixing, measured in a display at 620 nm. A comparison of the determinedabsorbency of samples and reference solution shows, under the describedconditions, the measured absorbency. TABLE 3 Comparison of theAbsorbency of Dextra Blueing before and after concentration Samples A₆₂₀Reference Solution 0.291 ± 0.012 (n = 8)  Samples after Concentration0.465 ± 0.020 (n = 88)

The absorbencies have increased by a factor of 1.6. That means, thatafter 45 minutes the volume of the samples is reduced by 37.5 μl, andindeed relatively uniformly, as may be seen by the distribution.

APPLICATION EXAMPLE NO. 5

For the dialysis of plasmid DNA, in a 96 well dialysis-plate, 110 μl ofthe samples having plasmid DNA pc DNA3.1hcSΔE44-N63(6.5 kb) was treatedagainst buffer Tris 10 mM pH 8 and subjected to shaking and mixing. Tothe samples, paranitrophenol (PNP) was added in the end concentration of978 μM and the reduction of the concentration was measured respectivelyafter two and four hours.

The content of DNA in the samples was likewise determined after two andfour hours in, respectively, eight parallel samples of a concentrationwith the aid of optical density. The detection was carried out at 400 nmfor PNP and 260 nm for the plasmid DNA.

The table below shows the balance of the plasmid-DNA: That is, theconcentrations were computed with a standard series of plasmids byoptical density.

Table: After 4-hour Output DNA Dialyzation, DNA in μg/ml in μg/ml  54.55 10 9.32 50 50.79

The balance of PNP in the samples, as per a standard series for thesamples where n=32: PNP after 2 hour PNP after 4 hour Item Output PNPDialysis Dialysis Concentration in the 978.6 38.96 0.56 sample in μMStandard deviation 1.01 4.6 0.60 in μM % from output 100 3.9 0.06Reference Numbers and Corresponding Components

-   1 Dialysis container-   2 Dialysate-   3 Sample plate-   4 Holder-   5 Well, (5 a conical tapered well)-   6 Material of the sample-   7 Dialysis membrane-   8 Magnetic stirrer-   9 Adhesive foil-   10 Float element-   11 Air escape opening-   12 Entry fitting (feed)-   13 Outlet fitting (exit)-   14 Float-   15 Float valve-   16 Float guide-   17 Pump-   18 Filter cartridge-   19 Line, for circulation of liquid-   20 Holder (FIG. 5)-   21 Shaker arm-   22 Shaker-   23 Receiving plate (receives contents of 3)-   24 Sealing means

1-35. (Cancelled)
 36. An apparatus for simultaneous dialysis of amultiplicity of liquid samples, the apparatus comprising: a dialysiscontainer adapted for receiving a dialysate therein, means for inflowand outflow of the dialysate, and a level controller; at least onesample plate disposable in the container for contact with the dialysate,the sample plate including a multiplicity of equal sample wells with a(n×8×12) matrix arrangement, wherein the wells define holding capacitiesfor microliter volumes, open upper ends, and lower ends closed by asemipermeable dialysis membrane lying in a plane, whereby the sampleplate does not protrude beyond the plane of the semipermeable membranein an area defined by the matrix arrangement, the sample plate furtherincluding means for releasing air imprisoned between the sample plateand the dialysate; means for holding the sample plate in contact with asurface of the dialysate; and means for moving at least one of thesample plate and the dialysate.
 37. The apparatus of claim 36, whereinthe sample wells are substantially cylindrical.
 38. The apparatus ofclaim 36, wherein the sample plate comprises a geometry of conventionalmicrotiter plates.
 39. The apparatus of claim 36, wherein at least someof the lower ends of the sample wells are respectively closed by singledialysis membranes.
 40. The apparatus of claim 36, wherein at least someof the lower ends of the sample wells are closed by at least one commondialysis membrane.
 41. The apparatus of claim 36, wherein the dialysismembrane comprises a plurality of membrane layers.
 42. The apparatus ofclaim 36, wherein the dialysis membrane is sprayed upon the sampleplate.
 43. The apparatus of claim 36, wherein the dialysis membrane isadhesively connected to an underside of the sample plate.
 44. Theapparatus of claim 36, wherein the dialysis membrane is at least one ofbonded, welded, and sprayed on an underside of the sample plate.
 45. Theapparatus of claim 36, wherein an adhesive foil is provided as areleasable closure of the upper ends of the sample wells.
 46. Theapparatus of claim 36, wherein a cover is provided as a releasableclosure of the upper ends of the sample wells.
 47. The apparatus ofclaim 36, wherein the dialysis container comprises at least one inletand at least one outlet, at least one of which is connected to a floatvalve.
 48. The apparatus of claim 47, further comprising means forremoval of substances to be dialyzed out of the dialysate, the removalmeans in fluidic communication with the at least one outlet and the atleast one inlet.
 49. The apparatus of claim 48, wherein the removalmeans comprises at least one ion exchanger.
 50. The apparatus of claim48, wherein the removal means comprises an absorber for bindingdetergents.
 51. The apparatus of claim 48, wherein the removal meanscomprises chelate forming substances.
 52. The apparatus of claim 47,wherein dialysate flow through the at least one inlet and through the atleast one outlet is adjustable.
 53. The apparatus of claim 52, whereinthe dialysate flow is adjustable by at least one of a valve and apositioning element.
 54. The apparatus of claim 47, wherein the dialysiscontainer, including the at least one inlet and the at least one outlet,forms at least a portion of a circulation system for the dialysate, thecirculation system further comprising a rotary pump.
 55. The apparatusof claim 36, wherein the sample plate is held in the dialysis containerby at least one stand element disposed on a bottom of the dialysiscontainer.
 56. The apparatus of claim 36, wherein the sample plate isheld by at least one or more float element disposed in the dialysatecontained in the dialysis container.
 57. The apparatus of claim 36,wherein the means for holding the sample plate includes means forproviding movement and mixing of a sample material contained in at leastone of the sample wells.
 58. The apparatus of claim 57, wherein themeans for providing movement and mixing comprises a shaker.
 59. Theapparatus of claim 57, wherein the means for providing movement andmixing comprises an ultrasonic source.
 60. The apparatus of claim 36further comprising at least one monitoring element for at least one ofmeasuring and treating the dialysate.
 61. The apparatus of claim 60,wherein the at least one monitoring element comprises means forsimultaneous measurement of conductivity.
 62. The apparatus of claim 60,wherein the at least one monitoring element comprises means forsimultaneous measurement of optical density.
 63. The apparatus of claim60, wherein the at least one monitoring element comprises a fluorescencedetector.
 64. The apparatus of claim 60, wherein the at least onemonitoring element comprises a thermometer.
 65. The apparatus of claim60, wherein the at least one monitoring element is provided for controlof the dialysis process.
 66. The apparatus of claim 36 furthercomprising means for transferring a sample from the sample plate. 67.The apparatus of claim 66, wherein the means for transferring the samplecomprises a receiving plate, which can be inverted and placed on thesample plate, the receiving plate including a plurality of sample wellswith a (n×8×12) matrix arrangement corresponding to the matrixarrangement of the sample wells of the sample plate.
 68. The apparatusof claim 67, wherein the means for transferring the sample comprisesusing centrifugation.
 69. The apparatus of claim 67 wherein the samplewells of the receiving plate define openings that correspondingly engageopenings defined by the sample wells of the sample plate to establish ashape fit.
 70. The apparatus of claim 69, wherein the sample wells ofthe sample plate comprise a conical shape and the openings of the samplewells of the sample plate are individually smaller than correspondingopenings of the sample wells of the receiving plate, where the openingscoincide when the receiving plate is set upon the sample plate.
 71. Theapparatus of claim 69, wherein the openings of the sample wells of thesample plate are individually substantially equal in size to theopenings of the sample wells of the receiving plate.
 72. The apparatusof claim 67, wherein the means for transferring a sample furthercomprises a seal between the sample plate and the receiving plate, theseal selected from at least one of a sealant and a paste.
 73. Theapparatus of claim 36, wherein the means for the moving the dialysatecomprises a magnetic stirrer.